What’s on the minds of groups involved in cell culture and fermentation in 2014? They want to build on recent achievements in reducing timelines and cost-of-goods (CoGs) while increasing efficiency and productivity by implementing potentially disruptive approaches and technologies. Those include genomic tools and synthetic biology, continuous bioprocessing, single-use applications, process intensification, raw-material integrity test methods, culture media optimization approaches, technology transfer, and small-scale models.
Raw Materials Matter: Culture media, supplements, and related ingredients are important raw materials in both cell culture and fermentation processes. Many speakers this year are focusing on the “nice-to-have” and “need-to-have” information from suppliers — who face some challenges in providing the data that users need.
In media and feed formulation optimization, many companies are revisiting the powder/liquid supply question. Determining and meeting quality standards involve analytical assays, formal risk analyses, and increased demands on purity, packaging, and the cold chain. Ingredients are screened using laboratory spectroscopy methods, for example, and statistical analysis. Differences in soy hydrolysates and trace-metal content, for example, have been shown to affect expression levels, harvest titers, and charge heterogeneity.
“As regulatory agencies increase their knowledge of biopharmaceutical manufacturing and variables that can affect performance,” writes Dave Kolwyck (principal scientist at Amgen) in the abstract for his Thursday afternoon talk, “their sensitivity to companies’ making changes in raw materials increases as well.” He will review technology developments that are changing the definition of comparability for raw materials and discuss how a risk-based approach can be applied to ensure consistency of manufacturing processes.
Single-Use Technology: Disposables are used in bioprocessing more every year. Single-use bioreactors eliminate some expenses associated with cleaning, assembly, operation, process validation, and equipment qualification. But many companies are facing difficulties with single-sourced consumables. The situation can cancel out some of the flexibility and interchangeability among existing systems, ultimately forcing users to rely on their vendor’s supply chain. Some speakers in the cell culture track will address these concerns.
Expansion and innovation continue in the single-use arena, however. In the abstract for his Thursday morning presentation, Berthold Boedeker (chief scientist at Bayer Healthcare) writes, “The ballroom concept and parallel operation of several products at a time is currently being evaluated as an alternative to the classical highly segregated good manufacturing practice (GMP) facility design for monoclonal antibodies (MAbs) and other biologics.” In his presentation, Boedeker will describe how advances in closed processing, disposables, and continuous processing can support such manufacturing approaches.
Blurring the Lines: Speaking of continuous bioprocessing, the lines between upstream and downstream are blurring somewhat. Although expression and harvest of a protein product stream will always mark the line between “production” and purification, the relative “batch” concepts of up- and downstream are changing.
Meanwhile, process development and optimization are closer to being concurrent activities than ever before. Modern modeling and scale methods allow companies to modify and monitor multiple process parameters and strategies: e.g., pH, feeding, base addition, initial seeding density, gassing, bioreactor geometry, and agitation.
BPI’s marketing and digital content strategist, Leah Rosin, conducted the following interviews as the conference program came together this summer. Participants addressed transient expression, a new single-use bioreactor technology, and how raw materials can affect product quality. Here, in Q&A format, is what they had to say.
Gavin Barnard, Yashas Rajendra (LillyResearch Laboratories)
Gavin Barnard (senior research scientist) and Yashas Rajendra (research scientist) are both in the biotechnology research discovery group at Lilly Research Laboratories. Barnard will be joining us for the “What’s Next in Biologics Production” session on Tuesday morning, 21 October 2014. His case study is titled “Development of Transient Expression System Based in CHO,” and it features unpublished data.
Abstract: We discuss the development of a polyethylenimine (PEI)-mediated transient CHO-GS KO system capable of generating high yields, scalable up to 2 L. This was achieved through rigorous optimization of cell density, DNA, and PEI amounts followed by process development strategies. The newly developed method increased our average expression yield by about fourfold relative to the existing transient HEK293 method.
What made you switch from using the HEK293? (Gavin) We still have the HEK293 system, which we use to support the bulk of our projects. We developed the transient Chinese hamster ovary (CHO) system as a complementary technology and alternative way of making molecules of interest.
Can you describe the method and equipment that you used to grow the transient CHO cells? (Yashas) It’s a shake-flask culture system for both growth and transfection of these cell lines. CHO transfection is a short, seven-day batch process. We are also evaluating stocks and maintenance in bioreactors.
How did you optimize cell densities within the system? (Yashas) High-cell density in combination with cell growth was our initial main strategy. So we chose to optimize transfection density between three and six million cells per milliliter. With a goal of the highest expression in the shortest timeframe, we found that a starting density of about four million cells was optimal.
What are the applications of transient expression in early process product development? How do you demonstrate comparability when moving to more traditional expression systems for full production? (Gavin) Our starting philosophy was to try to mimic what happens in late-stage development — and, optimally, manufacturing. Typically, what happens in our industry is that people use HEK293 cells early on as a fairly robust system to obtain significant amounts of protein early in discovery with minimal expenditure of time and resources. Ultimately, you have to reformat into CHO.
A couple of things happen in that conversion process. You are using a different cell line when you convert from HEK293 to CHO. You use different media. Sometimes you use different expression vectors and/or integration vectors. We approach the problem differently.
We looked at the final manufacturing process, which uses a stable cell line. And we tried to copy the conditions using the same process variables, the same media from the same manufacturers, and so on. Then we tried to develop a transient system — a quick system — that would minimize the number of variables we would need to change in the process.
That was our starting philosophy. Along the way, as we developed the system, we took a host of different molecules — both therapeutic molecules of interest and reagents — and we looked at product-quality attributes (PQAs). We looked at size, expression profile, and posttranslational modifications. In this process, we tracked those parameters, particularly for cases in which we have data. We expressed the same protein using a transient HEK293 system and a stable CHO line. And we found really good correlation between materials made from stable lines and from our transient CHO system.
What concerns or obstacles did your team face regarding purification of materials from high–cell-density cultures? (Gavin) We collaborated with our colleagues downstream in early stage development. Specifically, we pass on the material to them, and then they do the rigorous purification and analytical characterization. They actually did not encounter any purification obstacles, despite the fact that we are using CHO cells. We have fairly high cell viabilities; therefore, we don’t have some of the purification problems associated with growing to very high cell density. Culture media are essentially contaminated with dead cells and cell debris. We have not encountered that yet.
Finally, why are you attending the BPI Conference? (Gavin) The BPI Conference attracts a number of key participants in our industry. This gives us an opportunity to network with colleagues. We also believe that the method and technology we’ve developed here at Lilly are of use and of interest to the target audience.
Edward Chan (Genentech)
Edward Chan (technical specialist in the cell culture pilot plant at Genentech, a member of the Roche Group) will be joining us for the “Next Wave of Single-Use Manufacturing” session on Thursday morning, 23 October 2014. His presentation is titled “Unican: Dual Capability in Single Use Bioreactor.”
Abstract: A 200-L bottom-mounted single-use bioreactor was converted into a universal can, or “Unican,” which supports the use of single-use bioreactors with either bottom-mounted or top-mounted agitators. Implementation of the Unican increases flexibility for cell culture operations, improves assurance of supply, allows handling of any future bag film changes or issues, and increases the company’s ability to negotiate pricing with bag suppliers. As part of this project, mass transfer and cell culture experiments were performed to ensure optimal performance of both bags in the Unican. Cell culture data and mass transfer were comparable in both top-and bottom-mounted bags with historical data. Additionally, the system modification was performed such that no hardware requalification was required afterward. The system has been successfully implemented in a pilot plant for bioprocessing.
Why and where are single-use bioreactors used by Genentech in biopharmaceutical manufacturing? Single-use bioreactors have played a key role to allow Genentech power plants to meet increasing demands while maintaining a high success rate, reducing labor costs, increasing efficiency, and lowering the risk of contamination. They lower expenses associated with cleaning, maintenance, and operations, as well as equipment validation. In Genentech power plants, single-use bioreactors are used for phase 1, 2, and 3 development runs, IND toxicology studies, and transient transfections.
The UNICAN concept is interesting. Can you explain the problems with relying on a single vendor? What market instabilities in the supply chain led to this need? Typically, the consumables are designed to work with the hardware, which eliminates flexibility and interchangeability among existing systems. That forces end users to rely on a single vendor’s supply chain.
Unican adjusts such uncertainties arising from single-sourced bag supply. It increases a system’s applicability for good manufacturing practice (GMP) use through improved reliability and assurance of supply and enables options for addressing bag manufacturing changes.
Can you provide an overview of mass transfer in cell culture experiments you perform to validate the Unican system? We compared cell culture performance of multiple cell lines. Cell culture data and mass transfer from both the tops and bottoms of the bags were shown to be comparable with historical data.
What about tubing and connections and analytical instruments for process optimization? Have you had to change vendors for those? How do they work with the different bioreactor bags? No, we modified the system to allow bags from multiple suppliers to use the same hardware. Bag change-over requires under 30 minutes.
Last, why are you attending the BPI Conference? I want to share our work with the industry, and I want to learn from peers on ongoing work and advancement in single-use bioreactors.
Kenneth Green(Shire Pharmaceuticals)
Kenneth Green (head of manufacturing science and technology at Shire Pharmaceuticals) will be joining us for the “Impact of Raw Materials on Product Quality” session on Thursday afternoon, 23 October 2014. His presentation is titled “The Application of Risk Assessments to Identify and Mitigate Material Risks.”
Can you discuss some common risks associated with raw materials for cell culture? I categorize these into two areas. One category can affect performance: things that may be associated with undefined and defined materials used for cell culture media or feeds, as well as defined sources. The second category can compromise quality. For example, a contaminant might be introduced as part of a material supply chain or attributes associated with a particular material. Those can have a more serious impact on product quality.
What role can vendors play in risk management for raw materials? Vendors have an extremely important role. They can better manage the supply quality to determine whether variability is associated with materials they supply and then identify what that variability is and how to manage it from a quality perspective. And from a change-notification perspective, vendors can improve how they notify pharmaceutical companies when changes are made to raw materials. Some such changes could be deemed insignificant or small, yet still have an impact on the product or the process.
Can you describe how predictive modeling can aid in risk management for raw materials? This is an area that we’re very interested in because we collect a lot of data from incoming materials. Vendors typically supply data in certificates of analysis. And users (particularly at pharmaceutical companies) will do their own testing — particularly release testing and, in some cases, use testing. What we are trying to do is understand how we can better use those data to predict raw-material performance. One way to look at it is to use predictive modeling techniques and analysis to understand how the attributes of those materials will affect performance or product quality.
Why are you attending the BPI Conference? I want to learn what is current in the industry. I am very interested in how other companies are addressing issues with raw materials and how vendors are managing the supply of those materials from a quality perspective. I’m also interested in networking with colleagues, again to try to develop some industry-wide responses to some of the issues that we’ve been observing.